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Abstract Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder associated with features of accelerated aging. HGPS is an autosomal dominant disease caused by a de novo mutation of LMNA gene, encoding A-type lamins, resulting in the truncated form of pre-lamin A called progerin. While asymptomatic at birth, patients develop symptoms within the first year of life when they begin to display accelerated aging and suffer from growth retardation, and severe cardiovascular complications including loss of vascular smooth muscle cells (VSMCs). Recent works reported the loss of VSMCs as a major factor triggering atherosclerosis in HGPS. Here, we investigated the mechanisms by which progerin expression leads to massive VSMCs loss. Using aorta tissue and primary cultures of murine VSMCs from a mouse model of HGPS, we showed increased VSMCs death associated with increased poly(ADP-Ribosyl)ation. Poly(ADP-Ribosyl)ation is recognized as a post-translational protein modification that coordinates the repair at DNA damage sites. Poly-ADP-ribose polymerase (PARP) catalyzes protein poly(ADP-Ribosyl)ation by utilizing nicotinamide adenine dinucleotide (NAD+). Our results provided the first demonstration linking progerin accumulation, augmented poly(ADP-Ribosyl)ation and decreased nicotinamide adenine dinucleotide (NAD+) level in VSMCs. Using high-throughput screening on VSMCs differentiated from iPSCs from HGPS patients, we identified a new compound, trifluridine able to increase NAD+ levels through decrease of PARP-1 activity. Lastly, we demonstrate that trifluridine treatment in vivo was able to alleviate aortic VSMCs loss and clinical sign of progeria, suggesting a novel therapeutic approach of cardiovascular disease in progeria.

More effective approaches are needed in the treatment of blood cancers, in particular acute myeloid leukemia (AML), that are able to eliminate resistant leukemia stem cells (LSCs) at the bone marrow (BM), after a chemotherapy session, and then enhance hematopoietic stem cell (HSC) engraftment for the re-establishment of the HSC compartment. Here, we investigate whether light-activatable nanoparticles (NPs) encapsulating all-trans-retinoic acid (RA+NPs) could solve both problems. Our in vitro results show that mouse AML cells transfected with RA+NPs differentiate towards antitumoral M1 macrophages through RIG.1 and OASL gene expression. Our in vivo results further show that mouse AML cells transfected with RA+NPs home at the BM after transplantation in an AML mouse model. The photo-disassembly of the NPs within the grafted cells by a blue laser enables their differentiation towards a macrophage lineage. This macrophage activation seems to have systemic anti-leukemic effect within the BM, with a significant reduction of leukemic cells in all BM compartments, of animals treated with RA+NPs, when compared with animals treated with empty NPs. In a separate group of experiments, we show for the first time that normal HSCs transfected with RA+NPs show superior engraftment at the BM niche than cells without treatment or treated with empty NPs. This is the first time that the activity of RA is tested in terms of long-term hematopoietic reconstitution after transplant using an in situ activation approach without any exogenous priming or genetic conditioning of the transplanted cells. Overall, the approach documented here has the potential to improve consolidation therapy in AML since it allows a dual intervention in the BM niche: to tackle resistant leukemia and improve HSC engraftment at the same time.

Abstract The design of human model systems is highly relevant to unveil the underlying mechanisms of aging and to provide insights on potential interventions to extend human health and life span. In this perspective, we explore the potential of 2D or 3D culture models comprising human induced pluripotent stem cells and transdifferentiated cells obtained from aged or age-related disorder-affected donors to enhance our understanding of human aging and to catalyze the discovery of anti-aging interventions.

Abstract Cell-penetrating peptides (CPPs) are short chains of amino acids that have shown remarkable potential to cross the cell membrane and deliver coupled therapeutic cargoes into cells. Designing and testing different CPPs to target specific cells or tissues is crucial to ensure high delivery efficiency and reduced toxicity. However, in vivo/in vitro testing of various CPPs can be both time-consuming and costly, which has led to interest in computational methodologies, such as Machine Learning (ML) approaches, as faster and cheaper methods for CPP design and uptake prediction. However, most ML models developed to date focus on classification rather than regression techniques, because of the lack of informative quantitative uptake values. To address these challenges, we developed POSEIDON, an open-access and up-to-date curated database that provides experimental quantitative uptake values for over 2,300 entries and physicochemical properties of 1,315 peptides. POSEIDON also offers physicochemical properties, such as cell line, cargo, and sequence, among others. By leveraging this database along with cell line genomic features, we processed a dataset of over 1,200 entries to develop an ML regression CPP uptake predictor. Our results demonstrated that POSEIDON accurately predicted peptide cell line uptake, achieving a Pearson correlation of 0.87, Spearman correlation of 0.88, and r2 score of 0.76, on an independent test set. With its comprehensive and novel dataset, along with its potent predictive capabilities, the POSEIDON database and its associated ML predictor signify a significant leap forward in CPP research and development. The POSEIDON database and ML Predictor are available for free and with a user-friendly interface at https://moreiralab.com/resources/poseidon/ , making them valuable resources for advancing research on CPP-related topics. Scientific Contribution Statement: Our research addresses the critical need for more efficient and cost-effective methodologies in Cell-Penetrating Peptide (CPP) research. We introduced POSEIDON, a comprehensive and freely accessible database that delivers quantitative uptake values for over 2,300 entries, along with detailed physicochemical profiles for 1,315 peptides. Recognizing the limitations of current Machine Learning (ML) models for CPP design, our work leveraged the rich dataset provided by POSEIDON to develop a highly accurate ML regression model for predicting CPP uptake. Graphical Abstract

Abstract Advanced therapies with combined approaches of cell and nanomedicine‐based interventions are emerging. Corneal blindness with a severe form of limbal stem cell (LSC) deficiency is an example of defect with unmet clinical need. Towards cell‐based therapy, advanced enabling technologies are needed for efficient intracellular delivery of biomolecules both for in vitro disease modeling and for in vivo interventions. For this nanomedicine creates novel possibilities as light activatable polymeric nanoparticles (NPs) hold potential for controlled on‐demand cargo delivery with control of light. In this study, we used multi‐parametric surface plasmon resonance (MP‐SPR) technique in vitro for measuring NP intake in real‐time by therapeutic LSCs. Although a variety of NPs has been described, many challenges remain, and one is related to cell uptake of the formulations. Here, three different NP formulations were analyzed, one demonstrating clearly the highest cellular intake by LSCs. Importantly, data demonstrate that the labeling of NPs significantly reduce the cellular intake highlighting the importance of label‐free administration and quantification. The MP‐SPR based approach hold high potential as a powerful non‐invasive platform to be implemented in manufacturing of therapeutic cell‐ and cargo‐based interventions. For any therapy applications, validated non‐invasive and label independent NP intake measurement systems would be important.

Abstract Spatial control of gene expression is critical to modulate cellular functions and deconstruct the function of individual genes in biological processes. Light-responsive gene-editing formulations have been recently developed; however, they have shown limited applicability in vivo due to poor tissue penetration, limited cellular transfection and the difficulty in evaluating the activity of the edited cells. Here, we report a formulation composed of upconversion nanoparticles conjugated with Cre recombinase enzyme through a photocleavable linker, and a lysosomotropic agent that facilitates endolysosomal escape. This formulation allows in vitro spatial control in gene editing after activation with near-infrared light. We further demonstrate the potential of this formulation in vivo through three different paradigms: (i) gene editing in neurogenic niches, (ii) gene editing in the ventral tegmental area to facilitate monitoring of edited cells by precise optogenetic control of reward and reinforcement, and (iii) gene editing in a localized brain region via a noninvasive administration route (i.e., intranasal).

Endothelial cell (EC) activity is essential for tissue regeneration in several (patho)physiological contexts. However, our capacity to deliver in vivo biomolecules capable of controlling EC fate is relatively limited. Here, we screened a library of microRNA (miR) mimics and identified 25 miRs capable of enhancing the survival of ECs exposed to ischemia-mimicking conditions. In vitro, we showed that miR-425-5p, one of the hits, was able to enhance EC survival and migration. In vivo, using a mouse Matrigel plug assay, we showed that ECs transfected with miR-425-5p displayed enhanced survival compared with scramble-transfected ECs. Mechanistically, we showed that miR-425-5p modulated the PTEN/PI3K/AKT pathway and inhibition of miR-425-5p target genes (DACH1, PTEN, RGS5, and VASH1) phenocopied the pro-survival. For the in vivo delivery of miR-425-5p, we modulated small extracellular vesicles (sEVs) with miR-425-5p and showed, in vitro, that miR-425-5p-modulated sEVs were (1) capable of enhancing the survival of ECs exposed to ischemia-mimic conditions, and (2) efficiently internalized by skin cells. Finally, using a streptozotocin-induced diabetic wound healing mouse model, we showed that, compared with miR-scrambled-modulated sEVs, topical administration of miR-425-5p-modulated sEVs significantly enhanced wound healing, a process mediated by enhanced vascularization and skin re-epithelialization.

Tradicionalmente en la literatura especializada se reconocen modelos para la gestión de costos logísticos y la gestión de costos ambientales; pero no se evidencian propuestas teóricas que los integren. En este artículo se propone un modelo conceptual de gestión de costos logísticos ambientales en la cadena de suministros de combustibles y lubricantes, que tiene como propósito que en cada ciclo de gestión se logre la disminución progresiva de las pérdidas asociadas a derrames físicos y evaporaciones en la manipulación/conservación de dichos materiales. Se validaron sus componentes a partir de su funcionamiento en la cadena de suministros de combustibles y lubricantes, para lo que se utilizó un análisis factorial. Como principal resultado se presenta un modelo conceptual que sintetiza las áreas, variables, actores, la cuantificación de los costos en términos de eficiencia y pérdidas, entradas y salidas, de la gestión de costos logísticos ambientales en la cadena de suministros de estas sustancias.

The existence of a selective blood-brain barrier (BBB) and neurovascular coupling are two unique central nervous system vasculature features that result in an intimate relationship between neurons, glia, and blood vessels. This leads to a significant pathophysiological overlap between neurodegenerative and cerebrovascular diseases. Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease whose pathogenesis is still to be unveiled but has mostly been explored under the light of the amyloid-cascade hypothesis. Either as a trigger, bystander, or consequence of neurodegeneration, vascular dysfunction is an early component of the pathological conundrum of AD. The anatomical and functional substrate of this neurovascular degeneration is the BBB, a dynamic and semi-permeable interface between blood and the central nervous system that has consistently been shown to be defective. Several molecular and genetic changes have been demonstrated to mediate vascular dysfunction and BBB disruption in AD. The isoform ε4 of Apolipoprotein E is at the same time the strongest genetic risk factor for AD and a known promoter of BBB dysfunction. Low-density lipoprotein receptor–related protein 1 (LRP-1), P-glycoprotein, and receptor for advanced glycation end products (RAGE) are examples of BBB transporters implicated in its pathogenesis due to their role in the trafficking of amyloid-β. This disease is currently devoid of strategies that change the natural course of this burdening illness. This unsuccess may partly be explained by our misunderstanding of the disease pathogenesis and our inability to develop drugs that are effectively delivered to the brain. BBB may represent a therapeutic opportunity as a target itself or as a therapeutic vehicle. In this review, we aim to explore the role of BBB in the pathogenesis of AD including the genetic background and detail how it can be targeted in future therapeutic research.

Abstract The extracellular matrix (ECM) of engineered human cardiac tissues corresponds to simplistic biomaterials that allow tissue assembly, or animal derived off-the-shelf non-cardiac specific matrices. Decellularized ECM from human cardiac tissue could provide a means to improve the mimicry of engineered human cardiac tissues. Decellularization of cardiac tissue samples using immersion-based methods can produce acceptable cardiac ECM scaffolds; however, these protocols are mostly described for animal tissue preparations. We have tested four methods to decellularize human cardiac tissue and evaluated their efficiency in terms of cell removal and preservation of key ECM components, such as collagens and sulfated glycosaminoglycans. Extended exposure to decellularization agents, namely sodium dodecyl sulfate and Triton-X-100, was needed to significantly remove DNA content by approximately 93% in all human donors. However, the biochemical composition of decellularized tissue is affected, and the preservation of ECM architecture is donor dependent. Our results indicate that standardization of decellularization protocols for human tissue is likely unfeasible, and a compromise between cell removal and ECM preservation must be established in accordance with the scaffold’s intended application. Notwithstanding, decellularized human cardiac ECM supported human induced pluripotent-derived cardiomyocyte (hiPSC-CM) attachment and retention for up to 2 weeks of culture, and promoted cell alignment and contraction, providing evidence it could be a valuable tool for cardiac tissue engineering.

Although systemic inflammation and pulmonary complications increase the mortality rate in COVID-19, a broad spectrum of cardiovascular and neurological complications can also contribute to significant morbidity and mortality.The molecular mechanisms underlying cardiovascular and neurological complications during and after SARS-CoV-2 infection are incompletely understood. Recently reported perturbations of the epitranscriptome of COVID-19 patients indicate that mechanisms including those derived from RNA modifications and non-coding RNAs may play a contributing role in the pathogenesis of COVID-19.In this review paper, we gathered recently published studies investigating (epi)transcriptomic fluctuations upon SARS-CoV-2 infection, focusing on the brain-heart axis since neurological and cardiovascular events and their sequelae are of utmost prevalence and importance in this disease.

Ischemic stroke is becoming one of the most common causes of death and disability in developed countries. Since current therapeutic options are quite limited, focused on acute reperfusion therapies that are hampered by a very narrow therapeutic time window, it is essential to discover novel treatments that not only stop the progression of the ischemic cascade during the acute phase, but also improve the recovery of stroke patients during the sub-acute or chronic phase. In this regard, several studies have shown that endothelial progenitor cells (EPCs) can repair damaged vessels as well as generate new ones following cerebrovascular damage. EPCs are circulating cells with characteristics of both endothelial cells and adult stem cells presenting the ability to differentiate into mature endothelial cells and self-renew, respectively. Moreover, EPCs have the advantage of being already present in healthy conditions as circulating cells that participate in the maintenance of the endothelium in a direct and paracrine way. In this scenario, EPCs appear as a promising target to tackle stroke by self-promoting re-endothelization, angiogenesis and vasculogenesis. Based on clinical data showing a better neurological and functional outcome in ischemic stroke patients with higher levels of circulating EPCs, novel and promising therapeutic approaches would be pharmacological treatment promoting EPCs-generation as well as EPCs-based therapies. Here, we will review the latest advances in preclinical as well as clinical research on EPCs application following stroke, not only as a single treatment but also in combination with new therapeutic approaches.

Biomedical applications of nanoparticles require a fundamental understanding of their interactions and behavior with biological interfaces. Protein corona formation can alter the morphology and properties of nanomaterials, and knowledge of the interfacial behavior of the complexes, using in situ analytical techniques, will impact the development of nanocarriers to maximize uptake and permeability at cellular interfaces. In this study we evaluate the interactions of acrylamide-based nanogels, with neutral, positive, and negative charges, with serum-abundant proteins albumin, fibrinogen, and immunoglobulin G. The formation of a protein corona complex between positively charged nanoparticles and albumin is characterized by dynamic light scattering, circular dichroism, and surface tensiometry; we use neutron reflectometry to resolve the complex structure at the air/water interface and demonstrate the effect of increased protein concentration on the interface. Surface tensiometry data suggest that the structure of the proteins can impact the interfacial properties of the complex formed. These results contribute to the understanding of the factors that influence the bio-nano interface, which will help to design nanomaterials with improved properties for applications in drug delivery.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disease and smooth muscle cells are the most affected cells in HGPS individuals. Here, the authors report a microfluidics platform with HGPS induced pluripotent stem cells and show that inhibition of metalloprotease 13 may reduce smooth muscle cell loss.

Retinoids are involved in a wide range of cellular functions; as such, delivery systems for retinoids are of significant clinical interest. Here the authors review the advances in preclinical and clinical testing regarding retinoid formulations in the context of regenerative medicine, brain, cancer, skin and immune diseases.

In recent years, a great deal of effort has been undertaken with regards to treatment of pathologies at the level of the central nervous system (CNS). Here, the presence of the blood-brain barrier acts as an obstacle to the delivery of potentially effective drugs and makes accessibility to, and treatment of, the CNS one of the most significant challenges in medicine. In this Roadmap article, we present the status of the timeliest developments in the field, and identify the outstanding challenges and opportunities that exist. The format of the Roadmap, whereby experts in each discipline share their viewpoint and present their vision, reflects the dynamic and multidisciplinary nature of this research area, and is intended to generate dialogue and collaboration across traditional subject areas. It is stressed here that this article is not intended to act as a comprehensive review article, but rather an up-to-date and forward-looking summary of research methodologies pertaining to the treatment of pathologies at the level of the CNS.

The use of pharmacologically active compounds to manage and treat diseases is of utmost relevance in clinical practice. It is well recognized that spatial-temporal control over the delivery of these biomolecules will greatly impact their pharmacokinetic profile and ultimately their therapeutic effect. Nanoparticles (NPs) prepared from different materials have been tested successfully in the clinic for the delivery of several biomolecules including non-coding RNAs (siRNA and miRNA) and mRNAs. Indeed, the recent success of mRNA vaccines is in part due to progress in the delivery systems (NP based) that have been developed for many years. In most cases, the identification of the best formulation was done by testing a small number of novel formulations or by modification of pre-existing ones. Unfortunately, this is a low throughput and time-consuming process that hinders the identification of formulations with the highest potential. Alternatively, high-throughput combinatorial design of NP libraries may allow the rapid identification of formulations with the required release and cell/tissue targeting profile for a given application. Combinatorial approaches offer several advantages over conventional methods since they allow the incorporation of multiple components with varied chemical properties into materials, such as polymers or lipid-like materials, that will subsequently form NPs by self-assembly or chemical conjugation processes. The current review highlights the impact of high-throughput in the development of more efficient drug delivery systems with enhanced targeting and release kinetics. It also describes the current challenges in this research area as well as future directions.

Abstract Small extracellular vesicles (sEVs), through their natural ability to interact with biological membranes and exploit endogenous processing pathways to convey biological information, are quintessential for the delivery of therapeutically relevant compounds, such as microRNAs (miRNAs) and proteins. Here, we used a fluorescently‐labelled miRNA to quantify the efficiency of different methods to modulate the cargo of sEVs. Our results showed that, compared with electroporation, heat shock, permeation by a detergent‐based compound (saponin) or cholesterol‐modification of the miRNA, Exo‐Fect was the most efficient method with > 50% transfection efficiency. Furthermore, qRT‐PCR data showed that, compared with native sEVs, Exo‐Fect modulation led to a > 1000‐fold upregulation of the miRNA of interest. Importantly, this upregulation was observed for sEVs isolated from multiple sources. The modulated sEVs were able to delivery miR‐155‐5p into a reporter cell line, confirming the successful delivery of the miRNA to the target cell and, more importantly, its functionality. Finally, we showed that the membrane of Exo‐Fect‐loaded sEVs was altered compared with native sEVs and that enhanced the internalization of Exo‐Fect‐loaded sEVs within the target cells and decreased the interaction of those modulated sEVs with lysosomes.

Abstract Background Externally triggered drug delivery systems hold considerable promise for improving the treatment of many diseases, in particular, diseases where the spatial–temporal release of the drug is critical to maximize their biological effect whilst minimizing undesirable, off-target, side effects. Results Herein, we developed a light-triggerable formulation that takes advantage of host–guest chemistry to complex drugs functionalized with a guest molecule and release it after exposure to near infrared (NIR) light due to the disruption of the non-covalent host–guest interactions. The system is composed by a gold nanorod (AuNR), which generates plasmonic heat after exposure to NIR, a thin layer of hyaluronic acid immobilized to the AuNR upon functionalization with a macrocycle, cucurbit[6]uril (CB[6]), and a drug functionalized with a guest molecule that interacts with the macrocycle. For proof of concept, we have used this formulation for the intracellular release of a derivative of retinoic acid (RA), a molecule known to play a key role in tissue development and homeostasis as well as during cancer treatment. We showed that the formulation was able to conjugate approximately 65 μg of RA derivative per mg of CB[6] @AuNR and released it within a few minutes after exposure to a NIR laser. Importantly, the bioactivity of RA released from the formulation was demonstrated in a reporter cell line expressing luciferase under the control of the RA receptor. Conclusions This NIR light-triggered supramolecular-based modular platform holds great promise for theranostic applications.

The blood–brain barrier (BBB) is a dynamic interface responsible for maintaining the central nervous system homeostasis. Its unique characteristics allow protecting the brain from unwanted compounds, but its impairment is involved in a vast number of pathological conditions. Disruption of the BBB and increase in its permeability are key in the development of several neurological diseases and have been extensively studied in stroke. Ischemic stroke is the most prevalent type of stroke and is characterized by a myriad of pathological events triggered by an arterial occlusion that can eventually lead to fatal outcomes such as hemorrhagic transformation (HT). BBB permeability seems to follow a multiphasic pattern throughout the different stroke stages that have been associated with distinct biological substrates. In the hyperacute stage, sudden hypoxia damages the BBB, leading to cytotoxic edema and increased permeability; in the acute stage, the neuroinflammatory response aggravates the BBB injury, leading to higher permeability and a consequent risk of HT that can be motivated by reperfusion therapy; in the subacute stage (1–3 weeks), repair mechanisms take place, especially neoangiogenesis. Immature vessels show leaky BBB, but this permeability has been associated with improved clinical recovery. In the chronic stage (>6 weeks), an increase of BBB restoration factors leads the barrier to start decreasing its permeability. Nonetheless, permeability will persist to some degree several weeks after injury. Understanding the mechanisms behind BBB dysregulation and HT pathophysiology could potentially help guide acute stroke care decisions and the development of new therapeutic targets; however, effective translation into clinical practice is still lacking. In this review, we will address the different pathological and physiological repair mechanisms involved in BBB permeability through the different stages of ischemic stroke and their role in the development of HT and stroke recovery.

Introduction Ischaemic stroke is the most prevalent type of stroke and is characterised by a myriad of pathological events triggered by a vascular arterial occlusion. Disruption of the blood-brain barrier (BBB) is a key pathological event that may lead to fatal outcomes. However, it seems to follow a multiphasic pattern that has been associated with distinct biological substrates and possibly contrasting outcomes. Addressing the BBB permeability (BBBP) along the different phases of stroke through imaging techniques could lead to a better understanding of the disease, improved patient selection for specific treatments and development of new therapeutic modalities and delivery methods. This systematic review will aim to comprehensively summarise the existing evidence regarding the evolution of the BBBP values during the different phases of an acute ischaemic stroke and correlate this event with the clinical outcome of the patient.Methods and analysis We will conduct a computerised search on Medline, EMBASE, Cochrane Central Register of Controlled Trials, Scopus and Web of Science. In addition, grey literature and ClinicalTrials.gov will be scanned. We will include randomised controlled trials, cohort, cross-sectional and case-controlled studies on humans that quantitatively assess the BBBP in stroke. Retrieved studies will be independently reviewed by two authors and any discrepancies will be resolved by consensus or with a third reviewer. Reviewers will extract the data and assess the risk of bias of the selected studies. If possible, data will be combined in a quantitative meta-analysis following the guidelines provided by Cochrane Handbook for Systematic Reviews of Interventions. We will assess cumulative evidence using the Grading of Recommendations, Assessment, Development and Evaluation approach.Ethics and dissemination Ethical approval is not needed. All data used for this work are publicly available. The result obtained from this work will be published in a peer-reviewed journal and disseminated in relevant conferences.PROSPERO registration number CRD42019147314.

Caco-2 monolayers are a common in vitro model used to evaluate human intestinal absorption. The reference protocol requires 21 days post-seeding to establish a stable and confluent cell monolayer, which is used in a single permeability assay during the period of monolayer stability (up to day 30). In this work, we characterize variations in the tightness of the cell monolayer over the stable time interval and evaluate the conditions required for their re-use in permeability assays. The monolayer integrity was assessed through TEER measurements and permeability of the paracellular marker Lucifer Yellow (LY), complemented with nuclei and ZO-1 staining for morphological studies and the presence of tight junctions. Over 150 permeability assays were performed, which showed that manipulation of the cell monolayer in the permeability assay may contribute significantly to the flux of LY, leading to Papp values that are dependent on the sampling duration. The assay also leads to a small decrease in the cell monolayer TEER, which is fully recovered when cell monolayers are incubated with culture media for two full days. When this procedure is followed, the cell monolayers may be used for permeability assays on days 22, 25, and 28, triplicating the throughput of this important assay.

Stroke represents the second leading cause of mortality and morbidity worldwide. Ischemic strokes are the most prevalent type of stroke, and they are characterized by a series of pathological events prompted by an arterial occlusion that leads to a heterogeneous pathophysiological response through different hemodynamic phases, namely the hyperacute, acute, subacute, and chronic phases. Stroke treatment is highly reliant on recanalization therapies, which are limited to only a subset of patients due to their narrow therapeutic window; hence, there is a huge need for new stroke treatments. Nonetheless, the vast majority of promising treatments are not effective in the clinical setting due to their inability to cross the blood-brain barrier and reach the brain. In this context, nanotechnology-based approaches such as nanoparticle drug delivery emerge as the most promising option. In this review, we will discuss the current status of nanotechnology in the setting of stroke, focusing on the diverse available nanoparticle approaches targeted to the different pathological and physiological repair mechanisms involved in each of the stroke phases.

Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652.

Leukaemia cells resident in the bone marrow niche are often resistant to conventional therapies. In this study, the authors develop light-sensitive, polymeric, retinoic acid-containing nanoparticles that are able to modulate the differentiation of resistant leukaemia cells bothin vitro and in vivo.

Abstract Cardiovascular diseases are the main cause of death in the world and are often associated with the occurrence of arrhythmias due to disruption of myocardial electrical integrity. Pathologies involving dysfunction of the specialized cardiac excitatory/conductive tissue are also common and constitute an added source of morbidity and mortality since current standard therapies withstand a great number of limitations. As electrical integrity is essential for a well-functioning heart, innovative strategies have been bioengineered to improve heart conduction and/or promote myocardial repair, based on: (1) gene and/or cell delivery; or (2) conductive biomaterials as tools for cardiac tissue engineering. Herein we aim to review the state-of-art in the area, while briefly describing the biological principles underlying the heart electrical/conduction system and how this system can be disrupted in heart disease. Suggestions regarding targets for future studies are also presented.

Summary: During development, hematopoietic stem and progenitor cells (HSPCs) arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The genetic program driving human HSPC emergence remains largely unknown. We previously reported that the generation of hemogenic precursor cells from mouse fibroblasts recapitulates developmental hematopoiesis. Here, we demonstrate that human fibroblasts can be reprogrammed into hemogenic cells by the same transcription factors. Induced cells display dynamic EHT transcriptional programs, generate hematopoietic progeny, possess HSPC cell surface phenotype, and repopulate immunodeficient mice for 3 months. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by engagement of open enhancers and promoters, initiating silencing of fibroblast genes and activating the hemogenic program. However, GATA2 displays dominant and independent targeting activity during the early phases of reprogramming. These findings shed light on the processes controlling human HSC specification and support generation of reprogrammed HSCs for clinical applications. : Gomes et al. show that specification of hemogenesis in human fibroblasts is mediated by cooperative transcription factor binding. GATA2 displays dominance, interacts with GFI1B, and recruits FOS to open chromatin, simultaneously silencing the fibroblast program and initiating an endothelial-to-hematopoietic transition to definitive hematopoiesis. Keywords: hematopoietic stem cell, direct cell reprogramming, hemogenic reprogramming, cooperative binding, hemogenic endothelium, human endothelial-to-hematopoietic transition, hematopoietic transcription factor, GATA2, GFI1B, FOS

Rationale/aimDespite the increasing efficacy of recanalization therapies for acute ischemic stroke, a large number of patients are left with long-term functional impairment, devoid of efficacious treatments. CD34+ cells comprise a subset of bone marrow-derived mononuclear cells with the capacity to promote angiogenesis in ischemic lesions and have shown promising results in observational and in vitro studies. In this study, we aim to assess the efficacy of an autotransplant of CD34+ cells in acute ischemic stroke.Sample size estimates30 patients will be randomized for a power of 90% and alpha of 0.05 to detect a difference in 3 months infarct volume.Methods and designWe will screen 18–80 years old patients with acute ischemic stroke due to occlusion of a middle cerebral artery (MCA) for randomization. Persistent arterial occlusions, contra-indications to magnetic resonance imaging (MRI), premorbid dependency, or other severe diseases will be excluded. Treatment will involve bone marrow aspiration, selection of CD34+ cells, and their administration intra-arterially in the symptomatic MCA by angiography. Patients will be randomized for treatment at 7 (±2) days, 20 (±5 days) or sham procedure, 10 in each group.Study outcomesThe primary outcome will be infarct volume in MRI performed at 3 months. Secondary outcomes will include adverse events and multidimensional functional and neurological measures.Discussion/conclusionSTROKE34 is a PROBE design phase IIa clinical trial to assess the efficacy of intra-arterial administration of CD34+ cells 7 and 20 days after acute ischemic stroke.Trial registration (EU Clinical Trials Register)2017-002456-88.

There is a high quest for novel therapeutic strategies to enhance recovery after stroke. MicroRNA-124 (miR-124) has been described as neuroprotective and anti-inflammatory molecule. Moreover, miR-124 is a well described enhancer of adult neurogenesis that could offer potentially beneficial effects. Herein, we used miR-124-loaded nanoparticles (miR-124 NPs) to evaluate their therapeutic potential in an in vitro and in vivo model of stroke. For that, neuroprotective and neurogenic responses were assessed in an in vitro model of stroke. Here, we found that miR-124 NPs decreased cell death and improved neuronal differentiation of subventricular zone (SVZ) neural stem cell cultures after oxygen and glucose deprivation. In contrast, intravenous injection of miR-124 NPs immediately after permanent focal ischemia induced by photothrombosis (PT) did not provide a better neurological outcome. In addition, treatment did not affect the number of 5-bromo-2'-deoxyuridine (BrdU)- and doublecortin/BrdU- positive cells in the SVZ at the study endpoint of 14 days after PT. Likewise, the ischemic insult did not affect the numbers of neuronal progenitors in the SVZ. However, in PT mice miR-124 NPs were able to specifically augment interleukin-6 levels at day 2 post-stroke. Furthermore, we also showed that NPs reached the brain parenchyma and were internalized by brain resident cells. Although, promising in vitro data could not be verified in vivo as miR-124 NPs treatment did not improve functional outcome nor presented beneficial actions on neurogenesis or post-stroke inflammation, we showed that our NP formulation can be a safe alternative for drug delivery into the brain.

Inflammatory mechanisms triggered by microglial cells are involved in the pathophysiology of several brain disorders, hindering repair. Herein, we propose the use of retinoic acid-loaded polymeric nanoparticles (RA-NP) as a means to modulate microglia response towards an anti-inflammatory and neuroprotective phenotype (M2). RA-NP were first confirmed to be internalized by N9 microglial cells; nanoparticles did not affect cell survival at concentrations below 100 μg/mL. Then, immunocytochemical studies were performed to assess the expression of pro- and anti-inflammatory mediators. Our results show that RA-NP inhibited LPS-induced release of nitric oxide and the expression of inducible nitric oxide synthase and promoted arginase-1 and interleukin-4 production. Additionally, RA-NP induced a ramified microglia morphology (indicative of M2 state), promoting tissue viability, particularly neuronal survival, and restored the expression of postsynaptic protein-95 in organotypic hippocampal slice cultures exposed to an inflammatory challenge. RA-NP also proved to be more efficient than the free equivalent RA concentration. Altogether, our data indicate that RA-NP may be envisioned as a promising therapeutic agent for brain inflammatory diseases.

Minimizar a contaminação ambiental é um dos maiores desafios devido à forte pressão da sociedade por uma agricultura socialmente sustentável e ambientalmente segura. Objetivou-se com este trabalho determinar os parâmetros da tecnologia de aplicação visando o aumento da eficácia de aplicação, com segurança para o aplicador e o ambiente. Foram pulverizados três volumes de aplicação 40, 90 e 165 L ha-1, obtidos pela combinação de três velocidades (0,5; 1,5 e 3,0 Km h-1) e duas rotações do bocal pulverizador (210 e 315 rad s-1), sobre quatro etiquetas de papel hidrossensível cujas imagens foram digitalizadas e analisadas pelo programa computacional Image Tool. Além do espectro e densidade foram determinados os valores de porcentagem do volume acumulado de gotas com diâmetros menores que 100, 150, 200, 400 e 600 µm. A cobertura do alvo para o volume de aplicação de 40 L ha-1 atendeu às recomendações para o controle de gramíneas. Os volumes de aplicação de 40 e 90 L ha-1 apresentaram respectivamente 20,87 e 29,14 % do volume compreendido por gotas com diâmetros menores que 200 µm. Na aplicação de volume de pulverização de 165 L ha-1, obteve-se 44,29 % de volume acumulado de gotas com diâmetro superior a 600 µm.

The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young's modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.

In this study we demonstrate that CD34(+) cells derived from human embryonic stem cells (hESCs) have higher smooth muscle cell (SMC) potential than CD34(-) cells. We report that from all inductive signals tested, retinoic acid (RA) and platelet derived growth factor (PDGF(BB)) are the most effective agents in guiding the differentiation of CD34(+) cells into smooth muscle progenitor cells (SMPCs) characterized by the expression of SMC genes and proteins, secretion of SMC-related cytokines, contraction in response to depolarization agents and vasoactive peptides and expression of SMC-related genes in a 3D environment. These cells are also characterized by a low organization of the contractile proteins and the contractility response is mediated by Ca(2+), which involves the activation of Rho A/Rho kinase- and Ca(2+)/calmodulin (CaM)/myosin light chain kinase (MLCK)-dependent pathways. We further show that SMPCs obtained from the differentiation of CD34(+) cells with RA, but not with PDGF(BB,) can be maturated in medium supplemented with endothelin-1 showing at the end individualized contractile filaments. Overall the hESC-derived SMCs presented in this work might be an unlimited source of SMCs for tissue engineering and regenerative medicine.

In this study, we developed a methodology to improve the survival, vascular differentiation and regenerative potential of umbilical cord blood (UCB)-derived hematopoietic stem cells (CD34(+) cells), by co-culturing the stem cells in a 3D fibrin gel with CD34(+)-derived endothelial cells (ECs). ECs differentiated from CD34(+) cells appear to have superior angiogenic properties to fully differentiated ECs, such as human umbilical vein endothelial cells (HUVECs). Our results indicate that the pro-survival effect of CD34(+)-derived ECs on CD34(+) cells is mediated, at least in part, by bioactive factors released from ECs. This effect likely involves the secretion of novel cytokines, including interleukin-17 (IL-17) and interleukin-10 (IL-10), and the activation of the ERK 1/2 pathway in CD34(+) cells. We also show that the endothelial differentiation of CD34(+) cells in co-culture with CD34(+)-derived ECs is mediated by a combination of soluble and insoluble factors. The regenerative potential of this co-culture system was demonstrated in a chronic wound diabetic animal model. The co-transplantation of CD34(+) cells with CD34(+)-derived ECs improved the wound healing relatively to controls, by decreasing the inflammatory reaction and increasing the neovascularization of the wound.